Background Retinal and macular diseases are a major cause of untreatable vision impairment. Progressive neurodegenerative diseases, such as retinitis pigmentosa (RP), age-related Macular degeneration (AMD), and diabetic retinopathy have different etiology but ultimately lead to the death of rod and cone photoreceptors, the light sensitive neurons that initiate visual perception. Identification of disease causing mutations and elucidation of underlying mechanisms and pathways of photoreceptor degeneration constitutes the first step in developing targeted strategies for therapy. To identify genes involved in retinal degenerative diseases, we are performing linkage analysis in families and screening candidate genes for possible causative mutations. Furthermore, we are using mouse models of retinal disease to identify new genes that may cause retinopathies in humans. This is a powerful approach to uncover novel genes for human disease as well as to better understand the degenerative process in retinal neurons. Results 1. Linkage studies in human families Worldwide, RP is a major cause of inherited irreversible vision loss. It is clinically heterogeneous and characterized by night blindness, bone spicule-like pigmentation, and progressive constriction of visual fields. To date, 192 retinal disease loci have been mapped and 144 genes identified (see RetNet website). Mutations in at least 60 genes may cause RP;of these, 18 genes have been associated with ad forms of RP. Screening of the 18 disease genes has led to detection of mutations in 50%60% of adRP families;thus, genetic defects in many patients are yet to be identified. Through genotyping and linkage analyses, we have recently identified a BTB-Kelch protein, KLHL7, as a novel genetic cause of autosomal dominant retinitis pigmentosa (adRP). A large family of Scandinavian origin with adRP was genotyped and linkage analysis yielded a LOD score of 5 on chromosome 7p. A mutation screen of the genes in the critical interval revealed a mutation in KLHL7, a member of the Kelch-like family of genes. With our collaborators, we have confirmed that mutations in KLHL7 cause adRP in other families. On the basis of the presence of BTB and Kelch domains, we suggested and are currently exploring the possibility that KLHL7 participates as an adaptor and/or chaperone in the ubiquitin-proteasome protein-degradation pathway. We intend to characterize this gene, examine its interactors and generate a mouse model to investigate disease mechanisms and therapeutic options. This finding opens new research directions in our efforts to understand the molecular mechanisms of photoreceptors cell death in the degenerating retina. 2. Animal models of human retinal diseases We have recently elucidated the gene defects in several naturally occurring mutant mouse lines carrying retinal disease, including rd3, rd9, rd11, rd14 and rd16. At least three of these genes exhibit disease-causing mutations in patients with retinopathies (rd3, rd16, and rd9). Long-term studies have been initiated to profile gene expression in rod photoreceptors expressing GFP and flow-sorted from mice carrying rd mutations (e.g., rds, rd1, rd16). Several additional studies are summarized here: Two allelic lines of mice, rd11 and 2845, develop retinal degeneration early in life and can be useful models of human retinal disease. In collaboration with Dr Bo Chang (Jackson Labs), we performed a mutation screen of genes in the critical interval and identified a candidate gene mutated in both lines. Further characterization of the gene and its mutation is ongoing. Loss of function of Cep290 results in several forms of retinal degeneration. The mouse mutant, rd16, mimics human mutations in CEP290. CEP290, CC2D2a, and some BBSome members have emerged as major players in photoreceptor ciliogenesis. They are essential for photoreceptor development and their mutations are observed in patients suffering from syndromic ciliopathies, with early or late onset retinal dystrophy. The rd16 mutation is a hypomorphic allele characterized by rapid retinal degeneration with loss of photoreceptors and other milder sensory defects. We are applying genetic, biochemical, gene therapy, and cell biological approaches to determine how loss of Cep290 affects photoreceptor and retinal pigment epithelium development and function. By in vivo RNAi-mediated knockdown studies in the developing mouse retina we have identified and characterized a critical interactions of Cep290 with other ciliogenesis genes. To further elucidate the role of Cep290 in photoreceptor development and function and in retinal disease and to determine the domains of Cep290 important for specific intereactions, we have generated in collaboration with Lijin Dong (NEI, GEF), a null allele of Cep290, with lacZ knock-in. We have also generated a second hypomorphic allele of Cep290 using a genetrap construct from the Sanger Center. CEP290 interactions were further investigated and confirmed in co-injected zebrafish embryos in collaboration with Dr. Paul Lius laboratory (NHGRI) and in compound heterozygous mice. The Nrl-KO retina carries the unique phenotype of a cone-only retina. This retinal phenotype resembles those of late stages of AMD and cone-rod dystrophies, where loss of rod photoreceptors leaves a cone-only retina and is followed by cone degeneration. ERG, histology and IHC in Nrl-KO mice showed a rapid death of S- and M-cones between 2 to 4 month of age and stabilization thereafter. Microglia and Mller glia cells were activated starting at 2 months. Microarrays were performed and the data are being analyzed to identify gene misregulations that might trigger cone cell death (manuscript in preparation). Additional transgenic mouse lines displaying a degenerative phenotype have been identified in the past year in our colony. We are currently investigating the mutated gene for a new ADRP mouse line characterized by a flat ERG (cones and rods) at 4 months and normal ONL histology, albeit with no opsin expression detected by IHC. In addition, a spontaneous mutation in a transgenic line expressing Cre recombinase under control of the blue opsin promoter (BP-Cre) was noticed to cause a dramatic ERG phenotype that may prove to be a novel mouse model for the study of congenital stationary night blindness. Significance Our work in animal models of retinal degeneration has highlighted an interesting convergence on microtubules and biogenesis and transport functions associated with primary cilia. These results will have significance for treating patients with Leber congenital amaurosis (LCA) and other forms of retinal degeneration. Furthermore, we have highlighted novel molecular pathways (e.g. protein degradation and lipid metabolism), which we are exploring to gain deeper insights into the mechanisms of retinal degeneration.